This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The cell cycle helps to regulate cell growth by maintaining constant volumes and homeostasis. Gap junctions (GJs) are an integral part of this process because it is partially through intercellular communication that cells help to maintain uniform cytosolic metabolite and ion concentrations. Mitosis, an integral part of the cell cycle, causes significant morphological and biochemical changes throughout the entire cell. Certainly, some of the biggest changes in GJ structure morphology and distribution occur as tissue culture cells internalize their GJs as they go into mitosis and undergo cell rounding and cytokinesis. In unstimulated (normally trafficking cells) NRK cells, Cx43 isolated from immunoprecipitated cell lysates show three bands on Western blots. These include a nonphosphorylated form (NP) and two phosphorylated forms (P1 and P2) that are predominately phosphorylated on multiple, unidentified serine sites. GJ plaques contain predominantly the P1 and P2 forms while the NP form is localized intracellularly. Using immunofluorescence, nocodazole synchronized mitotic cells display an entirely intracellular localization of Cx43. The Cx43 found in these mitotic cells is uniquely phosphorylated and migrates as a distinct P3 species in a p34cdc2/cyclin B kinase-dependent manner. Most connexins contain sites for multiple sites for phosphorylation. These phosphorylation sites are typically found in the C-terminal region, although Cx56 has an additional phosphorylation site located in the cytoplasmic loop. These phosphorylation sites are important for proper protein trafficking, assembly and degradation and likely play a role in signal transduction as these connexins contain several protein kinase consensus phosphorylation sequence. Nonetheless, the carboxyl terminus can be deleted and GJs will still assemble so the significance of phosphorylation sites in assembly is not yet understood. Treatment of cells released from nocodazole block with proteosomal, lysosomal and protein synthesis inhibitors demonstrated that protein degradation plays a role in the disappearance of the P3-Cx43 after mitosis. While substantial protein synthesis occurs during reformation of GJs after mitosis, there is intriguing evidence from live cell imaging that some connexin recycling may also be occurring. At the light microscopy resolution level, there is no morphology information on the nature of connexin bearing structures since the intracellular localization appears as small bright spots and old versus new protein cannot be distinguished from each other. This project is focused on determining whether recycling occurs during mitosis and uses the tetracysteine technology to do so.